This is the second installment of our series on the state of biohacking. You can read the first installment “Safari in the Biohacking Society” and watch for more articles in the future.

Bioprint Is the New Print

The greatest bridge between the world of makers and the world of biohackers is probably the “mighty” 3D printer. What if instead of using plastics, we could use any biomaterial to build three-dimensional structures? And what if we could use a special ink, let’s call it a “bioink”, made of living cells to print messages or patterns?

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When BioCurious Started Bioprinting

Our 2014 safari sponsored by Paris Biohackerspace lead us to BioCurious: a mandatory stop among biohacker communities in North America. This pioneering biohackerspace hosts a number of great people collaborating on a DIY bioprinter project. Their bioprinting adventure started in 2012, when they had their first meet-ups. Patrik D’haeseleer, who is currently leading the project with Maria Chavez, gave us some details and updates on their story.

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At that time, they were looking for community projects that could bring new people in and let them collaborate on a project right away. None of them had a passion for a specific bioprinting application, nor did they have previous knowledge on how to build this kind of printer. Still, it appeared to be a fairly approachable technology that people could play with. You don’t even necessarily need a wet lab to start. It’s a project that has something for everyone, whether it’s 3D printing, inkjet printing, 3D design, electronics, Arduino, or cell culture  —  you name it! Everyone has something to learn or something to teach.

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Printing by Trial and Error

(Or the importance of publishing what went wrong with your experiments.)

“You can just take a commercial inkjet printer. Take the inkjet cartridges and cut off the top essentially. Empty out the ink and put something else in there. Now you can start printing with that,” Patrik explained.

That’s how they started at BioCurious. They printed with arabinose as ink, which is a natural plant sugar, on big coffee filters as paper. Then they put that filter paper on a culture of bacteria called E.Coli in a Petri dish. Those bacteria are commonly used in biolabs, but that time they were genetically modified to make them produce a green fluorescent protein in the presence of arabinose. At the end, the cells started to glow exactly where arabinose was printed.

The technique was actually feasible, even if not perfect. But that’s not how the team get this exciting pattern visible under UV light : “I ♥ BIOCURIOUS”.

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Using a commercial printer is too limiting. “You may need to reverse engineer the printer driver or disassemble the paper handling machinery in order to be able to do what you want,” explains Patrik. So the group at BioCurious decided to build their own bioprinter from scratch. This second version is the one you can find on Instructables.

Thanks to the Hackteria collective and GaudiLabs, our biohackers had the idea to use stepper motors from CD drives to build a 2D platform. Add an inkjet cartridge as a print head and interface it with a compatible open source Arduino shield, and you have your own DIY bioprinter for $150!

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The next and still current challenge deals with the consistency of the ink. The inkjet commercial ink cartridges work essentially with an ink that is pretty watery. But bioink implies a more gel-like material with high viscosity. The DIY BioPrinter group has been experimenting with different syringe pump designs that could allow them to inject small amount of viscous liquid through the print head.

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And Now Comes the 3D

Starting with an already existing 3D platform seemed like the best way to go beyond 2D patterns. The DIY group tried to turn their existing 3D printer into a bioprinter, by putting a bioprinting head on it in the first place. Soon, they were halted by the fact that working on a commercial machine would require some difficult reverse engineering and software modification to perfect the process… After a couple of months, this led to a dead end.

RepRap is the main open source family of 3D printers. After buying the most feasible and affordable open source printer as a kit, they just had to switch out the plastic extruding print head for a needle or a set of needles. This bioprint head is connected with flexible tubes to the syringe pumps, which can stay stationary.

‘’The RepRap community is really what has made the whole 3D printing revolution possible” said Patrik.

Soon enough there was a community around 3D bioprint tinkering at home and in biohackerspaces such as BioCurious, BUGSS and Hackteria, all sharing their experiements.

Working with Life

The Holy Grail of bioprinting is printing 3D organs for transplants (we will come back to this in our next installment). Working with human or mammalian cells is complex. You would need to have someone in the lab every day taking care of the cells and to keep everything as sterile as possible. Therefore, the current long-term project of the group is essentially a proof of concept that they can make a functional plant organ and get it to photosynthesize. This will be an artificial leaf!

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There is not that much work done on plant cells. Therefore, there are a lot of open science questions raised. You need to figure out what kind of cell types you will use, how to connect them together, what a 3D structure of a leaf looks like etc. According to Patrik, 3D printing with plant cells fit much better for DIY community lab than mammilian cells.

Whether it works or not, the interest here is to test things and see how they grow. A commercial application is not the only purpose for biohackers, even though some scientists are a bit overwhelmed by the potential of their research.

“We are not very goal oriented, like we want to make a startup out of bioprinting and sell a product, make millions of dollars… There are not too many plants in desperate need of leaf transplants! We participate in this project because it’s a fun thing to do. We make some progress week after week,” comments Patrik.

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3D Bioprinting with Plant Cells Raise Challenges

The first step is to figure out the material in which the cells are going to be resuspended. A matrix material may hold the cells in place until they grow and make connections. Some current experiments at BioCurious use a gel-like material called alginate, which has very interesting properties. Sodium alginate is soluble in water, but viscous whereas calcium alginate solidifies instantaneously. It reminds me of spherification techniques that you see in food science, where a solid droplet is full of liquid on the inside.

Several syringe pump designs are in testing now, but all are tested with the same comparison: one syringe pump containing the cells within an alginate solution, and the second one with calcium chlorite. When the two materials come in contact, the structure solidifies. Then you actually print a solid with embedded cells. Optimization is in progress.

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The second challenge is about the cell type needed. “Should we differentiate all the cells first and print the cells where we think they should go? Should we print undifferentiated cells and growth factors at the same time to let them differentiate and rearrange in situ?” The question is still open for Patrik. The DIY group experimented with diverse cell types and did not recommend using carrot cells as people usually do. These stem cells are undifferentiated, which means they can give rise to different cell types under good conditions, but they are often contaminated.

Now, have a look to the following recipe: “Take a leaf. Smash it up to get single cells. Resuspend them into alginate. Print with this solution plus the medium the plant cells need, the whole thing being extruded into a calcium chlorite bath. Over the first weeks, the cells started to bleach. But eventually, the green comes back. The explanation could be a transient dedifferentiation of the cells at first and then a return to a leaf’s cell’s state.” Leaf cells from tobacco plants, a very robust and short time generation lab model, seem to be promising!

3D Printing Outside BioCurious: Other Exciting Ideas

BUGSS – Baltimore

Baltimore Underground Science Space is currently building a platform call 3DP.BIO that aims to connect scientists, engineers, and designers to accelerate research and development. They focus on resin printers, developing the control software and a biocompatible resin that can be used to make 3D scaffolds for cell growth.

Mediated Matter MIT Medialab

The MIT Medialab has also been tinkering with biomaterials and printing technologies. Here is Markus Kayser in Mediated Matter’s lab, who is tinkering with weird sea animals, pilling dried crab shells and mixing it to create structures made from biomaterials.

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Also in his lab, people can 3D print structures using silk worms.

Here is an interview, directed by Neri Oxman, with Sunanda Sharma, a member of the Mediated Matter Lab who is explaining some of their projects and perspectives around chitin as biomaterial.

JUICY PRINT at London Biohackspace
Here is an example where you still print with some biomaterials, except this time the material has been genetically modified like so: “JuicyPrint is like a 3D printer that can be fed with fruit juice and can be used to print out useful shapes made of bacterial cellulose, a strong and exceptionally versatile biopolymer.” The bacteria Gluconacetobacter hansenii, which uses fruit juice as a food source, are genetically modified in order to make them unable to produce cellulose under a light stimulation. Therefore only bacteria in the dark patches will produce cellulose. Then, the structure of the final product can be manipulated by shining a new pattern of light onto the culture.

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Another way to grow tissues or organs would be to use an already existing 3D structure as a scaffold for cells.

image courtesy of Pelling Labs

image courtesy of Pelling Labs

PELLING Lab: Making Ears Out of Apples
Here is a protocol from Andrew Pelling in which “you slice an apple, wash it in soap and water, then sterilize it. What’s left is a fine mesh of cellulose into which you can inject human cells — and they grow.”

Counter Culture labs

Why 3D print when you can use already shaped forms? Look at this surprising example below of a pig heart at Counter Culture Labs, a biohackerspace in Oakland.

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Here they strip out all the cells from a donor organ which was a pig heart, leaving only the connective tissue to make it a ghost organ. Then, the idea would be to repopulate it with someone’s cells.

Let’s now jump to next installment with more biodesigned material and architecture.

Another way to think about 3D Bioprinting and biomaterials is to actually let nature grow by itself… Can you guess what the picture below is and where in the world this lab is based?